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You searched for: EV200090 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200090 1/2 Homo sapiens Cell culture supernatant (d)(U)C
SEC commercial
UF
Arab, Tanina; Mallick, Emily 2021 67%

Study summary

Full title
All authors
Tanina Arab, Emily R. Mallick, Yiyao Huang, Liang Dong, Zhaohao Liao, Zezhou Zhao, Olesia Gololobova, Barbara Smith, Norman J. Haughey, Kenneth J. Pienta, Barbara S. Slusher, Patrick M. Tarwater, Juan Pablo Tosar, Angela M. Zivkovic, Wyatt N. Vreeland, Michael E. Paulaitis, Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesi (show more...)We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
promonocytic line U937
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC commercial
UF
Protein markers
EV: CD81/ TSG101/ CD63/ CD9
non-EV: BiP/GRP78/ GM130
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
promonocytic line U937
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
TH-641
Pelleting: speed (g)
110000
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ CD81
Not detected EV-associated proteins
TSG101/ CD9
Not detected contaminants
BiP/GRP78/ GM130
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
CD81
Not detected EV-associated proteins
CD9/ CD63
Detected EV-associated proteins
CD63/ CD81
Detected EV-associated proteins
CD9/ CD81/ CD63
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
25-250
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.00E+09
EM
EM-type
Transmission-EM
Image type
Wide-field
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV200090 2/2 Homo sapiens Cell culture supernatant (d)(U)C
SEC commercial
UF
Arab, Tanina; Mallick, Emily 2021 67%

Study summary

Full title
All authors
Tanina Arab, Emily R. Mallick, Yiyao Huang, Liang Dong, Zhaohao Liao, Zezhou Zhao, Olesia Gololobova, Barbara Smith, Norman J. Haughey, Kenneth J. Pienta, Barbara S. Slusher, Patrick M. Tarwater, Juan Pablo Tosar, Angela M. Zivkovic, Wyatt N. Vreeland, Michael E. Paulaitis, Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesi (show more...)We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
T lymphocyte line H9
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC commercial
UF
Protein markers
EV: CD81/ TSG101/ CD63/ CD9
non-EV: BiP/GRP78/ GM130
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
T lymphocyte line H9
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
TH-641
Pelleting: speed (g)
110000
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
BiP/GRP78/ GM130
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
CD81
Not detected EV-associated proteins
CD63
Detected EV-associated proteins
CD81/ CD63
Detected EV-associated proteins
CD9/ CD81/ CD63
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
25-250
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.00E+09
EM
EM-type
Transmission-EM
Image type
Wide-field
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200090
species
Homo sapiens
sample type
Cell culture
cell type
promonocytic
line U937
condition
Control condition
Control condition
separation protocol
(d)(U)C
SEC commercial
UF
(d)(U)C
SEC commercial
UF
Exp. nr.
1
2
EV-METRIC %
67
67